A total of 38 samples (Blood, lung exudates, swab from trachea and larynx) were collected from dead birds that were suspected to be the case of Mycoplasma or E. coli infections during November, 2008 to June, 2009. Ten (10) samples were collected from apparently healthy chickens at regular slaughter that assumed to be free from chronic respiratory diseases clinically as control. The samples that were confirmed as Mycoplasma or E. coli or mixed infection by culture and PCR were subjected for cytological evaluation. Gross pathological examination was conducted on necropsy. Tissues from lungs, liver, trachea, heart and spleen organs were collected at necropsy and were preserved as per requirement. Part of the samples was fixed in 10% neutral buffered formalin for histopathological examinati on as per earlier described procedure (Luna 1968). Clean glass slides were gently touched on to the lesion of lung and immediately lifted off and air dried, stained with Giemsa staining and observed under a microscope using a 100× magnification with oil immersion. The counting was done gradually from one side to another side and numbers of inflammatory cells were estimated by counting 300 cells. Only Mycoplasma, E. coli or complicated Mycoplasma infected cases determined on necropsy findings were considered under this count.
Lung and tracheal swabs were collected either in nutrient or Mycoplasma broth supplemented with supplement G (Oxoid, Milan, Italy) and kenamycin. The nutrient broth containing suspected swab was incubated in aerobic condition at 37°C for 48 h. Upon growth of the organism, 100 μl of inoc ulums (infected media) were inoculated into EMB agar plate (Oxoid, Milan, Italy). Plates were incubated at 37°C for 48 h. After 48h, production of typical metallic sheen in EMB agar was considered as E. coli infection . Mycoplasma broth (Oxoid, Milan, Italy) containing suspected swab was incubated at 37°C for 72 h. After growth, 100 μl of Inoculum (infected media) were inoculated into Mycoplasma agar (Oxoid, Milan, Italy) with 20% of equine serum and plates were incubated for 10 da ys at 37°C in humid chamber with 10% CO2 . After 2 or 3 passages Mycoplasma , when present, produced typical colony with fried egg appearance.
Polymerase chain reaction (PCR) was used to detect the organisms following the procedure described by Lauerman et al., (1995). Commercial PCR kit ( PCR Master Mixture Kit, GeNeiTM , Banglore Genei, BDA Industrial Suburb, Peenya, India) was used for this purpose. Mycoplasma gallisepticum vaccine; MG TS- 11(Merial Select, Inc, Gainesville, USA) was used as positive control for Mycoplasma gallisepticum during PCR. Wizard R Genomic DNA Purification Kit (Promega Corporation. 2800 Woods Hollow Road. Madison, USA) was used to extract the DNA from Mycoplasma vaccine and Mycoplasma isolates as per manufacturer’s instructions. Primers and thermal profile were adopted from previous work (Lauerman et al., 1995). DNA amplification was performed in an oil-free thermal cycler (Master Cycler Gradient, Eppendorf, Germany). The amplified products were analyzed by 1.5% agarose gel (Promega Corp. Madison, WI, USA). The gel was visualized under UV light on a trans illuminator (Labortechnik, Germany). The expected product size of M. gallisepticum and M. synoviae were of 900 and 497bp, respectively.
Cell counting results obtained from blood of Mycoplasma and E. coli infected birds and healthy control birds were anal ysed with Bonferroni joint confidence interval, Mann-Whitney U test (Johnson and Wichern, 2002) using the software MS Excel, SPSS and MINITAB 13.